Tissue chamber

ABSTRACT

The present invention relates to systems and methods for tissue processing and analysis. Tissue chambers are configured to allow single-container chemical processing, imaging, and wax embedding of tissue samples in a single container without manipulation between steps. Tissue chambers with features to support the tissue sample and allow fluid flow between the tissue sample and the tissue chamber surface are disclosed. The features may be index matched to sample structures of interest or dissolvable in clearing solution to allow for in-chamber imaging with minimal distortion. Specialized tissue processing and wax removal apparatuses are also disclosed including for use with tissue chambers having frangible portions to permit ease of wax removal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalApplication No. 62/674,911, filed May 22, 2018, the contents of whichare incorporated by reference.

FIELD OF THE INVENTION

The present disclosure relates generally to histological systems andmethods for simplified positioning, chemical processing, and/or imagingof tissue samples including imaging, staining, fixing, wax embedding,and wax removal in a single chamber.

BACKGROUND

Histology and histopathology involve the study of cells and tissuesunder a microscope to diagnose and monitor diseases, such as cancer.Many of the fundamental techniques involved in histological analysis area century or more old and histological analysis is primarily performedby trained medical professionals.

For standard histological methods, such as creating a digitalhistological image, current steps include placing a tissue sample insidea plastic cassette with perforated walls that allow fluid access andexposing the cassette to a fluid environment that changes compositionover time to provide chemical fixation of the tissue. The tissue iseventually dehydrated and permeated with wax before removing thewax-embedded specimen from the cassette by melting the wax. For each ofthe processing steps, the tissue sample is generally resting on thefloor and/or wall of a plastic cassette providing poor fluid access tothose portions of the sample without agitation.

After the wax permeation and removal, the specimen is re-positioned inmolten wax and allowed to cool again to fix the specimen in anorientation that permits sectioning slices of the specimen in a plane ofchoice, optimized for clinical interpretation. The slices are thenplaced on a slide, stained, and then either viewed directly by apathologist or presented to an imager for digitization.

The above approach requires manipulation of tissue during processing,incurring labor costs and significant processing time. Furthermore,small specimens including small skin biopsies and biopsies from thegastrointestinal tract where orientation is critically relevant mayrotate and bend freely while the cassette is submerged in fluid andexposed to agitation or flow. Those specimens can therefore requiresignificant manipulation and processing time for histological analysis.

Additionally, the full suite of processing steps described above must becompleted before a pathologist can begin substantive analysis of thespecimen thereby incurring labor costs and delaying diagnoses.

SUMMARY

The present invention provides a single chamber solution for sampleprocessing (e.g., dehydration, fixation, staining, and wax embedding)thereby reducing costs, labor, and time required for histologicalanalysis. Furthermore, the present invention allows for the efficientincorporation of intermediary imaging steps during tissue processingthereby offering benefits such as earlier access to diagnosticinformation and the potential to avoid the costly steps of manualcutting, staining, and slide distribution where the need for suchactivities can be ruled out based on initial imaging results.

Single chamber solutions provided herein allow a user to initiallyorient a sample within the chamber in the desired position for imagingand/or sectioning and then perform all sample processing includingfixation and/or dehydration, initial dyeing, imaging, and eventual waxembedding without having to touch or otherwise manipulate the sampleagain. Accordingly, tissue chambers of the invention allow for automaticsample processing using various processing devices described herein.Container aspects such as features on the container surface to allow forfluid access, exchange, and/or flow to all sides of the sample, allowfor the sample to be successfully processed (e.g., dehydrated, fixed,stained, cleared, and wax embedded) without the need for agitation ormanipulation of the sample. Sealable ports, such as “self-sealing”syringe injection ports, permit fluid exposure, motion, and exchangewhile preventing potential air-bubble formation and trapping that couldaffect imaging. Index matched or solvent-susceptible features allow forinitial sample imaging through the features without significantdistortion. Additionally, inclusion of a substantially non-fluorescentand non-reflective sponge for mounting and/or positioning helps ensureposition is maintained, prevents artifacts of tissue compression, allowsfluid flow around specimen, and provides a surface for improved‘wetting’. When adequately index matched and optically transmissive, itcan also allow imaging through more than one surface, of particularvalue for use of the multiphoton modality known as second harmonicgeneration.

Alternatives have been explored for fixing the position of smallspecimens during tissue processing so that re-positioning at thewax-embedding step is not necessary such as the techniques described inU.S. Pat. No. 8,796,038 and US 20080227144, incorporated herein byreference. However, none of those prior methods allow for imaging afterthe clearing step, will typically not work for orienting all types ofbiopsies including gastrointestinal or skin biopsy specimens, and stillneed eventual removal by manual cutting and staining prior to visualinterpretation or digital scanning.

The present invention provides a significant advantage over existingtechniques by allowing a specimen to be placed in a container forhistologic analysis before chemical processing (e.g., fixation, orexposure to a dehydration solution) and to undergo chemical processingin the oriented position in a single container device that can be usedfor all the steps of dehydration, staining, clearing, and imaging.Furthermore, tissue chambers of the invention can then be used forwax-embedding and automated or machine-assisted removal of thewax-embedded sample using a wax removal device configured to work withtissue chambers of the invention.

Single-chamber processing as described herein can provide the additionaladvantage of minimizing reagent use. As noted, tissue chambers caninclude features operable to minimize the contact area between the floorand/or walls of the chamber and the sample, thereby permitting goodfluid access to all areas of the sample for fixatives, stains,dehydration solutions, wax, and/or other processing fluids. The walls ofthe chamber as well as the features can be optically clear and/orrefractive index matched to the clearing solution and/or the structuresof the sample to be examined (e.g., organelles or proteins). In certainembodiments, the features may comprise a material that dissolves in thepresence of certain solutions used in sample processing (e.g., aclearing solution) such that the features space the sample from thevessel walls and provide good fluid contact to all portions of thesample for processing but have dissolved before any initial imaging ofthe sample within the chamber and therefore do not disrupt the imagingprocess. The walls of the tissue chamber itself (or imaging windowportions therein) are substantially optically clear and/or index matchedto the clearing solution and/or the structures of the sample to beexamined in preferred embodiments.

Processing of the sample within the chamber in a fixed position forimaging allows for tight control over the volume of the chamber and thereagents used in processing. Instead of placing a cassette containingtissue that is open to a fluid environment that may be hundreds of timesthe volume of the tissue as in prior techniques, a single sampleprocessing vessel as described herein can have minimal dead-volume,reducing expenditure on reagents. Reagent conservation is particularlyimportant for controlling dye costs, which could otherwise beprohibitive, particularly for fluorescent markers. Hence, asample-tailored reagent vessel is particularly suited for processingthat incorporates dyeing of un-embedded specimens, and especiallyfluorescent dyeing where the cost of the dye may be the largestcost-component.

The single chamber techniques described herein also reduce processingtimes over existing methods. With current methods, it is more economicalto wait until sufficient samples have been received and “grossed”(placed into cassettes) before loading a tissue processor. The singlechamber approach can be combined with a specialized tissue processoroperable to receive tissue chambers of the present disclosure through,for example, interfacing with fluid inlet/outlets of the chamber. Once achamber is loaded with a specimen, the specimen can be plugged in andprocessed immediately; reducing the time the specimen must sit idlewaiting for additional samples as in multiplex processing. The sameapplies to the steps subsequent to embedding including slide collation,slide staining, and organization and scanning of slides fordigitization.

As noted above, single chamber systems described herein allow forvarying geometries to more tightly correspond to individual samplegeometry, reducing chamber dead volume and reagent consumption. Chamberscan be designed so that the sample is matched to the minimum-size vesselaccommodating the specimen. For example, a long core biopsy can beplaced in a long thin channel. In certain embodiments, the chamber has ageometry that is approximately that of a thick common microscope slide,with dimensions of approximately 2.5 mm×75 mm×10 mm.

Vessels can be coded (e.g., with a machine-readable symbol such as amatrix barcode (QR) or UPC code, or any symbol of recognizable shape,color, or reflective pattern) to provide sample and patientidentification and to allow a tissue processor to automaticallyrecognize the geometry of the tissue chamber being used and to adjustinput volumes accordingly, thereby further minimizing wasted reagents.In various embodiments, the vessel itself may be color coded to indicategeometry. Similarly, microscope scanning and imaging time can also bereduced with sample-specific sized chambers. While microscope slidescanners use various approaches to minimize slide scanning time, theyare still inefficient and manually dependent to varying extents. Currentsystems typically take a low power image and use image processingalgorithms to estimate the position and size of the tissue, but they areadversely affected by difficult-to-control artifacts such as mountingvariability, dirt, and smudges. As a result, manual supervision isrequired to ensure tissue is not missed and that large empty spaces arenot imaged. Operator adjustment of scanning area is a time-consumingcomponent of slide scanning which can result in repeat scans and highaverage slide scan times.

Coded, sample-size specific chambers as described herein can help avoidmanual interventions while maximizing efficiency for image scanning.Because a tissue is placed in the smallest chamber in which it fits andthe imager is able to read the chamber coding to determine the size ofthe region to scan, the entire possible tissue location region isimaged. The chance for error can be reduced and operator interventionshould not be required, providing efficient imaging with reduced laborcosts and errors.

Tissue chambers may include a trough area or cavity cutaway sized tocontain a sample and various processing fluids wherein processing of thesample occurs. The trough may be in fluid communication with one or morefluid inlets or outlets operable to provide processing fluids to thetrough. The tissue chamber may comprise additional material surroundingthe trough to allow for ease of manipulation and/or orientation of thechamber within various processing apparatuses. Tissue chambers caninclude one or more locating members such as a post configured to fitwithin a corresponding recess in various processing apparatuses tothereby locate the tissue chamber relative to fluid inlets/outlets,imaging objectives, wax removal tools, or other items. In variousembodiments, the locating member may be on the processing apparatus andthe tissue chamber may comprise a corresponding recess to receive themember.

Tissue chambers may comprise a frangible area in the floor or walls ofthe trough to provide a controlled separation of the floor or wall fromthe remainder of the tissue chamber upon the application of sufficientforce thereto. Such a frangible area may comprise a region of thinner orweaker material and can allow for efficient machine separation of anoriented, wax-embedded sample from the tissue chamber.

Tissue chambers can include wax-retention members within the trough suchas barbs configured to stabilize the wax-embedded sample within thetrough during processing.

Aspects of the invention include a container for holding a tissue samplewith the container comprising a cavity or trough for receiving a tissuesample and a wall having an interior surface adjacent to the cavity andan exterior surface. In some embodiments, the chamber is loaded on theimaging side and an optically transparent imaging cover or sheet may beaffixed after tissue loading in such a manner that the tissue is noteasily moveable in any direction while rendering the chambersubstantially sealed to liquid and air. In another embodiment the tissuecan be loaded into a cavity on the surface opposite the imaging surfaceand adjacent to the underside of the imaging surface. A cover can beplaced over the cavity after the tissue is loaded in order to render thetissue substantially immovable within the chamber and to substantiallyseal the chamber to liquid or air. The wall may include an opticalwindow with the window comprising a plurality of features on theinterior surface configured to contact the tissue sample and permitfluid flow between the tissue sample and the window. In some embodimentsthe surface opposite the optical window (e.g. is also opticallytransparent or transmissive of specific wavelengths and may be used asan imaging window. The transmissive wavelengths may be thosecorresponding to precisely half the wavelength (twice the frequency) ofthe excitation laser wavelength, as would be generated by secondharmonics (second harmonic generation).

The optical window can include a refractive index approximately equal toa refractive index of a fluid in which the tissue sample is immersedprior to imaging (e.g., a clearing solution). The refractive index ofthe clearing solution or other fluid to be used in processing the tissuesample may be approximately equal to the refractive index of a structureof the tissue sample to be analyzed. The optical window can comprise arefractive index of about 1.5 to about 1.7.

The size of the chamber may be any size which accommodates a tissuesample for microscopic analysis. In various embodiments the externalplanar dimensions of the chamber are approximately those of a microscopeslide, generally approximately 2.5 mm×75 mm or 1 inch by 3 inches. Theimaging portion of the chamber may be substantially smaller in someembodiments. For example, the imaging portion of a chamber for prostatecore biopsies may be between about 1 mm×15 mm and about 3 mm×40 mm,thereby fitting in a chamber with the external dimensions of a standardmicroscopy slide. In some embodiments the planar dimensions may belarger so as to accommodate specific specimen types having dimensionslarger than the above-referenced core biopsies. For example, for imagingof an eye enucleation specimen, the chamber may have dimensions ofbetween about 25 mm×25 mm and about 50 mm×50 mm. In other embodiments,the imaging chamber dimensions can be large enough to accommodatespecimens typically referred to as large format histology specimens, or“whole mounts”, which are in the range of about 65 mm×50 mm in planardimension. Similarly, the imaging chamber height may be any heightrequired for accommodating a specific specimen type. For example,heights may be anywhere between about 200 μm and about 15 mm. In someembodiments, the imaging chamber may be between about 200 μm and 500 μmin height, best suited for cytology specimens and very small biopsies.In other embodiments the chamber height may be between about 500 μm and1.5 mm, typically best suited for small and core biopsies.

Incorporation of a sponge support as discussed above may require ataller chamber height to accommodate both the sponge and the specimen.For example a chamber incorporating a sponge support may have a heightof between about 1 mm and about 3 mm for small and core biopsies. Inother embodiments, the chamber height can be between about 3 mm andabout 6 mm, which may be best suited for regular tissue sections. Instill other embodiments, the chamber height can be between about 6 mmand 15 mm, dimensions which may accommodate large format histologyspecimens as well general intermediate to large un-sectioned samples.

The external dimensions of a container comprising the chamber can varydepending on the dimensions of the enclosed tissue chamber and will belarge enough to allow for any required fluid channels or external portplugs as described herein. In some embodiments the external dimensionsof the container may include a height between about 1 mm and 10 mm. Inother embodiments the container height may be between about 500 um and 1mm. In other embodiments the container height can be between about 10 mmand 20 mm.

The plurality of features may comprise a material having a refractiveindex approximately equal to a refractive index of a fluid in which thetissue sample is immersed prior to imaging (e.g., a clearing solution).In certain embodiments, the refractive index of the clearing solution orother fluid to be used in processing the tissue sample may beapproximately equal to the refractive index of a structure of the tissuesample to be analyzed.

The plurality of features can comprise a material having a refractiveindex of about 1.5 to about 1.7. The plurality of features may comprisea material having a refractive index of between about 1.53 and about1.60. The plurality of features can comprise a material that dissolvesin the presence of an organic solvent. The organic solvent may be aclearing solution such as benzyl alcohol and benzyl benzoate (BABB).

In various embodiments, the container may comprise a porous compressiblematerial configured to contact the tissue sample on a side opposite theoptically clear window. In some embodiments the porous compressiblematerial is a plastic sponge. The sponge cell size may be any size thatenables adequate tissue support with minimal compression and may beanywhere in the range of 10 μm to 5 mm. The sponge cell size may be in arange that helps wet both the tissue and optical surfaces without airbubble trapping. In preferred embodiments, the sponge cell size isbetween 50 and 500 μm when dry. In other preferred embodiments thesponge cell size is between 50 and 200 μm. The sponge may be open cellor closed cell. In preferred embodiments the sponge is open cell. Inpreferred embodiments the sponge is substantially non-fluorescent. Insome embodiments the sponge is fabricated from a material that has arefractive index between about 1.45 and about 1.7. In some embodimentsthe sponge has a refractive index of between about 1.53 and 1.60. Thesponge material may be selected to approximately match the refractiveindex of the cleared tissue sample to be imaged.

The container may comprise one or more fluid ports in fluidcommunication with the cavity for receiving the tissue sample and aspace outside the chamber. In preferred embodiments the chamber containstwo ports. The two ports may be on the same surface, facilitatingconnection to a fluid exchange system or processor. In preferredembodiments the fluid ports are self-sealing, such as with a rubberizedor silicone plug or surface that permits introduction of a needle butwhich seals upon needle removal. In some embodiments the self-sealingports are needle-free connectors, such as those that include aself-closing valve that opens when a tube connector is attached.

The container can comprise a material that is resistant to acid, amaterial that is resistant to organic solvents such as BABB, a materialthat is resistant to alcohols and/or a material that is resistant totemperatures up to about 75 degrees Celsius. The sponge can comprise amaterial that is resistant to acid, a material that is resistant toorganic solvents such as BABB, a material that is resistant to alcoholsand/or a material that is resistant to temperatures up to about 75degrees Celsius.

In some embodiments, the cavity for receiving tissue samples maycomprise a frangible area. The frangible area can be located at aperimeter of the wall of the cavity. The frangible area may comprise anarea of thinned material relative to a remainder of the wall of thecavity.

The container may comprise one or more wax-retention members extendingfrom the interior surface of the wall and/or one or more locatingmembers extending from the exterior surface of the wall.

Aspects of the invention may include a method for analyzing a tissuesample including steps of orienting a tissue sample in a tissue chamberin a desired position; exposing the tissue sample to a first solutionfor chemical processing in the desired position in the tissue chamber;exposing the tissue to a fluid in which the tissue sample is immersedprior to imaging (e.g., a clearing solution) in the desired position inthe tissue chamber; imaging the tissue sample in the desired position inthe tissue chamber; and/or wax embedding the tissue sample in thedesired position in the tissue chamber.

The clearing agent may be BABB. The first solution may comprise adehydrant, a fixative, a dye, and/or some combination thereof includingwhere the fixative may be a dehydrant. In certain embodiments, the dyemay be a fluorescent dye and the imaging step can comprise fluorescentimaging. The desired position may be a desired position for sectioningof the wax-embedded tissue sample and or imaging of the tissue sample.The tissue chamber can comprise a plurality of features disposed on aninterior surface of the tissue chamber and configured to contact thetissue sample and permit fluid flow between the tissue sample and theinterior surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a tissue chamber having a trough and fluid inlets andoutlets.

FIG. 2 shows a top view of a tissue chamber having wax-retentionmembers.

FIG. 3 shows a cutaway view of a tissue chamber with locating membersand wax-retention members.

FIG. 4 shows a tissue chamber having a plurality of features for spacinga sample away from the chamber wall.

FIG. 5 shows a wax removal device in the open position.

FIG. 6 shows a wax removal device in the open position with a tissuechamber loaded therein.

FIG. 7 shows a wax removal device in the wax cutting position.

FIG. 8 shows a wax removal device in the wax removal position.

FIG. 9A shows a cutaway view of a wax removal device in the openposition with a tissue chamber loaded therein.

FIG. 9B shows a cutaway view of a wax removal device in the wax cuttingposition.

FIG. 9C shows a cutaway view of a wax removal device in the wax removalposition.

FIG. 10 illustrates a container 1001 including a support sponge 1013according to certain embodiments.

FIG. 11 illustrates some of the internal configuration of the container1001 shown in FIG. 10.

DETAILED DESCRIPTION

The present invention provides apparatuses, systems, and methods for thevisual histologic analysis of tissue during chemical processing (e.g.,fixing, dehydrating, dying, and staining) and wax embedding whilereducing manual intervention, human contact, and labor costs duringprocessing. Systems and methods allow for initial placement of a tissuesample in a single container in a preferred orientation for waxembedding and sectioning and/or imaging. The tissue sample can then bechemically processed (fixed, dehydrated, and/or dyed) and wax embeddedin the single container without subsequent repositioning. Furthermore,the tissue sample can be dyed, cleared and imaged intact to provide aninitial pathological analysis potentially negating the need forcontinued expensive processing, embedding, sectioning, staining, andanalysis. Systems and methods of the invention allow for simple machineseparation of the wax encased sample ready for sectioning in amicrotome.

FIG. 1 shows a tissue chamber 101 having a trough 107 for receiving andprocessing a tissue sample. Samples may be obtained, for example, duringsurgery, biopsy, fine needle aspiration, culture, or autopsy and arepreferably obtained for histological analysis. Tissue chambers 101and/or troughs 107 therein may be provided in a variety of sizes and mayinclude a mark 109 (human and/or machine-readable) that can correspondto the trough 107 or chamber 101 size and/or provide informationregarding the subject from which the sample was obtained, the type ofsample, and/or the type of analysis to be performed. Once read by amachine or human, the mark 109 may be used to tailor tissue processing(e.g., reagent selection, reagent volumes, or processing apparatusselection and configuration) and/or to label imaging data.

Tissue chambers 101 may include a remainder area surrounding the trough107 to increase the overall size and allow for ease of manipulation.Cutaways 103 or openings in the chamber 101 can reduce the mass of thechamber 101 along with reducing the required material in production,time of production, and the associated costs thereof. One or more fluidinlets/outlets 105 are in fluid communication with the trough 107 and anoutside surface of the chamber 101. The fluid inlets/outlets 107 caninterface with the corresponding fluid inlets/outlets in variousprocessing apparatuses to provide and remove processing fluids such asfixatives, dehydrating fluids, stains/dyes, clearing solution, or waxfor embedding.

The walls of the tissue chamber or relevant portions thereof (e.g., animaging window) may be optically clear and/or index matched to theclearing solution and/or the sample structures to be measured. Thetissue chamber 101 is thereby operable to contain a tissue sample forall processing steps for histological analysis while allowing forperiodic imaging of, for example, an intact and wax-free sampleincluding fluorescent dye-based imaging techniques. After dying, fixing,dehydrating, and/or any other processing steps are performed, wax can beintroduced to the trough 107 via the fluid inlets/outlets 105 to providea wax-embedded sample in a block of wax ready for sectioning andsubsequent analysis.

Accordingly, a tissue sample can be initially oriented within the trough107 in the desired position for both initial imaging and latersectioning and then left untouched throughout the remainder of theprocessing, imaging, wax embedding and removal steps.

Tissue chambers may be constructed of materials such as metals,plastics, a cyclic olefin polymer, or glass. Preferably the chambermaterial does not react with the tissue sample or any of the processingsolutions with which its surfaces come in contact. Chambers can beconstructed of multiple materials in certain embodiments. For example,the trough may be constructed of an unreactive and index matchedmaterial but, to reduce costs, the remainder of the chamber may beconstructed of a different, cheaper material.

FIG. 2 shows a top view of a tissue chamber 201 with fluidinlets/outlets 205, cutaways 203 and a trough 207. Within the trough 207may be wax-retention members 209 such as barbed posts configured tolocate and retain hardened wax and a sample held therein to a floor ofthe trough 207. The trough 207 can include a frangible area 211 on itsfloor consisting of, for example, an area of thinner or weaker materialsuch that, upon application of a shear force between the floor of thetrough 207 and the remainder of the chamber 201, the floor of the trough207 will separate from the remainder of the chamber 201 along the linesdefined by the frangible area 211. The frangible area 211 can be sizedand located to comprise the floor of the trough 207 and thewax-retention members 209 such that, upon separation along the frangiblearea 211, a wax-encased sample, coupled to the floor of the trough 211can be removed from the remainder of the chamber 201 for furtherprocessing (e.g., sectioning in a microtome). The wax-retention members209 can be spaced in a manner such that relevant samples can be fitbetween them in the trough 207 if necessary.

FIG. 3 shows a cutaway view of a tissue chamber 301 with fluidinlets/outlets 305 shown providing fluid access to the trough 307 fromthe outside surface of the chamber 301. The trough 307 compriseswax-retention members 309 as well as a frangible area 311 as describedabove. The bottom surface of the trough 307 can comprise locatingmembers 313 such as posts or tabs (or corresponding recesses forreceiving such members). The locating members 313 may correspond tocomplimentary locating recesses on the surface of various processing andimaging apparatuses. It will be readily apparent that while describedherein with respect to the members being present on the chamber 301 andthe corresponding recesses being present on the apparatuses, the reversearrangement would also provide the same function. The locating members313, when positioned in their corresponding recesses, may serve tolocate the chamber 301 and the trough 307 within the apparatusesrelative to, for example, a fluid coupling for the fluid inlets/outlets305, a wax-cutting blade, a plunger for separating the trough 307 flooralong the frangible area 311, an imaging objective, a light source, orvarious other processing tools.

In certain embodiments, the locating members 313 are attached to thefloor of the trough 307 and remain so after separation at the frangiblearea 311 resulting in a wax-embedded sample, in a wax block secured tothe now separated trough 307 floor by wax-retention members 309 andlocatable by locating members 313 protruding from the surface of thetrough floor 307 opposite the surface retaining the wax-embedded sample.The locating members 313 can therefore be used to locate the wax blockcontaining the sample for subsequent processing for example in amicrotome for sectioning. Tissue chambers may be reusable or single-useitems. For example, frangible tissue chambers are generally consideredsingle-use items.

FIG. 4 shows a tissue chamber 401 having a plurality of features 403 forspacing a sample away from the chamber wall 405. Spacing the sample awayfrom the otherwise flat surface of the chamber wall 405 allows forprocessing solutions such as dehydrating, fixing, clearing, and dyesolutions, to access all sides of the sample. In the absence of suchfeatures 403 the sample would rest against the flat surface of thechamber wall 405 sealing it off from the fluids and increasingprocessing times, reducing processing effectiveness (and subsequentanalysis quality), and/or requiring manipulation or agitation tore-orient the sample and expose the obstructed surfaces to the fluids.Features may be of any shape including cones, pyramids, needles,cylinders, spheres, cubes, ridges, spikes, or other 3-dimensionalshapes. Features may include porous structures or recesses in a materialsurface to allow fluid penetration or access. As the features 403 aredesigned to reduce surface area contact between the sample and thevessel or chamber wall 405, shapes such as cones or pyramids thatprovide a large base area in contact with the chamber wall 405 with aminimal contact point at the top supporting the sample are preferable.Features 403 should be shaped and spaced such that they provide theminimal contact surface area with the sample while still supporting thesample above the surface of the wall 405 and enough weight distributionso as not to puncture or otherwise penetrate the sample.

The features should have a height or depth sufficient to allow fluid toflow between the supported sample and the surface of the chamber wall.In various embodiments, features may have a height or depth about 1 μmto about 5 mm.

An apparent drawback to such features 403 would be their deleteriouseffects on imaging quality. Accordingly, in various embodiments thefeatures may be constructed of a material similar to the wall 405 of thechamber 401 and be index matched to the clearing solution and/or thesample structures to be examined. The features 403 will thereby provideminimal distortion during imaging. In other embodiments, the features403 may be constructed of a material different from the walls 405 of thechamber 401 and that material may be configured to dissolve in thepresence of one or more of the processing solutions (e.g., the clearingsolutions). Because the clearing solution is generally applied beforeimaging, if the features 403 dissolve in the presence of the clearingsolution, they will not be present to distort the subsequent imaging.Clearing solutions may comprise benzyl alcohol and benzyl benzoate(BABB) and, accordingly, features 403 may comprise materials known todissolve in BABB.

Processing devices of the invention may include wax removal devicesoperable to manipulate tissue chambers described herein. Such devicescan comprise a base with a spring-loaded platform on which the tissuechamber may be placed. There may be a hole in the middle of the platformto accommodate a plateau shaped to match the base of the sample troughthat includes positioning holes that match the positioning posts on thebottom of the tissue chamber. When pressed from above, the tissuechamber can be lowered on the spring loaded platform such that thecentral plateau presses up against the bottom of the sample trough,breaking it along the thinned perimeter and thus releasing the samplefrom the chamber.

The wax removal device can also include a central piston that holds twoknives pointing down towards the ends of the sample trough from above.When lowered, these knives cut into the wax, separating the portion ofwax within the sample trough from the wax that extends into the fluidinlet and outlet.

The wax removal device may be operable through three positions (actuatedmachine or manually via a handle). An open position, in which the arm israised, may lift both the central piston and the larger chamber pistonabove the base enough to enable placement of the tissue chamber onto thebase. A wax cutting position can be where the pistons have been loweredin unison by the arm to the point where the knives in the central pistonhave cut through the wax. Stops on the guide rails of the base canprevent the central piston from descending any further into the tissuechamber base. A wax removal position, where the arm is lowered furthersuch that the larger chamber piston has pushed the tissue chamber downcan force the sample trough base to break free of the tissue chamber. Aspring mechanism on the insert that connects the handle to the centralpiston can enable the movement to the wax removal position while thecentral piston remains still, pressed against the stops.

After moving to the wax removal position and breaking the sample trough,the arm may be raised back to the open position and thesample/wax/trough base removed. The sample would then be ready forplacement into a microtome for cutting.

FIGS. 5-8 show a wax removal device according to certain embodiments ofthe invention and configured to work with tissue chambers describedherein. Wax removal devices are useful for removing a wax embeddedsample from a tissue chamber after wax embedding. FIG. 5 illustrates awax removal device 501 in an open position with the handle 503 in araised position providing access to the spring-loaded platform 507 forthe placement of a tissue chamber 509 thereupon. The spring-loadedplatform 507 consists of an outer portion, the size and shape of whichgenerally conforms to the dimensions of the tissue chamber 509 placedthereupon and is configured to support said tissue chamber 509 frombelow. The spring-loaded platform 507 also comprises an inner portion,the size and shape of which generally conform to the dimensions of afrangible trough portion of the tissue chamber 509 containing thewax-embedded sample 511.

The inner portion is solidly supported from below while the outerportion may be supported from below by springs or be otherwise capableof being depressed below the level of the central portion in response toa downward force on the spring-loaded platform 507.

The handle 503 is operably associated with a central portion 513 havingwax-cutting blades 517 at the end proximal to the spring-loaded platform507. The handle 503 is operable to apply downward force on the centralportion 513 and, accordingly, the wax-cutting blades 517 toward thespring-loaded platform 507 and a tissue chamber 509 placed thereupon.The tissue chamber 509, the spring-loaded platform 507, and thewax-cutting blades 517 are located relative to each other such that whenthe handle 503 is operated, the wax-cutting blades 517 are forced intoand through the wax in the tissue chamber 509 to cut the wax-embeddedsample 511 out from surrounding wax in the tissue chamber 509. The waxremoval device 501 comprises stops 505 operable to limit the downwardmotion of the central portion 513 and the associated depth reached bythe wax-cutting blades 517 so that the wax-cutting blades 517 cut onlythrough wax inside the tissue chamber 509 but do not cut through thefloor of the tissue chamber 509.

The handle 503 is also operably associated with an outside portion 515having a plunger 523 at the end proximal to the spring-loaded platform507. While the stops 505 limit the downward motion of the centralportion 513, the outside portion 515 is able to continue its downwardmotion in response to further operation of the handle 503. The plunger523 generally conforms to dimensions of the outer portion of the springloaded-platform 507 and comprises an opening that generally conforms insize and shape to the dimensions of the central portion of thespring-loaded platform 507. Accordingly, when forced down into contactwith a tissue chamber 509 on the spring-loaded platform 507, the plunger523 applies downward pressure only to the outer portion of thespring-loaded platform which in turn is depressed below the level of therigidly supported inner portion. The inner portion thereby applies anupward force to the frangible trough portion of the tissue chamber 509containing the wax-embedded sample 511 while the plunger applies adownward force to the remainder 525 of the tissue chamber 509surrounding the trough portion. These opposing forces create a shearforce at the thinned or otherwise frangible area such that the frangiblearea breaks, freeing the cut wax-embedded sample 511 from the tissuechamber 509.

Accordingly, full motion of the handle 503 is operable to move both thecentral portion 513 and outside portion 515 downward toward thespring-loaded platform 507. The wax-cutting blades 517 cut the waxsurrounding the wax-embedded sample 511 and are stopped while theoutside portion 515 and associated plunger 523 continue downward,breaking the remainder 525 of the tissue chamber away from the cutwax-embedded sample 511 and pushing the remainder 525 and the outerportion of the spring-loaded platform 507 down below of the level of thenow separated was-embedded sample 523 which can then be removed from thewax removal device 501 for further processing.

FIG. 6 shows the wax removal device 501 in the open position with atissue chamber 509 positioned on the spring-loaded platform 507. Thehandle 503 is still in the raised position.

FIG. 7 shows the wax removal device 501 in the wax cutting positionwhere the handle 503 has been partially operated such that the stops 505are acting on the central portion 513 and the wax-cutting blades 517have cut the wax surrounding the wax-embedded sample 511 but the plunger523 has not broken the frangible area of the tissue chamber 509.

FIG. 8 shows the wax removal device 501 in the wax removal positionwhere the handle 503 has been fully operated such that the outsideportion 515 has forced the plunger 523 downward, breaking the remainder525 of the tissue chamber downward on the spring-loaded platform 507 andapart from the now separate wax-embedded sample 511.

FIGS. 9A-9C show a cutaway illustration of a wax removal deviceaccording to certain embodiments of the invention. The central portion513 and outside portion 515 of the wax removal device 501 and theirrespective association with the wax-cutting blades 517 and the plunger523 are shown. The cut away illustrations further show the inner andouter portions of the spring loaded platform 507 and the function of thewax removal device 501 to cut out the wax-embedded sample 511 andseparate it from the remainder 525 of the tissue chamber for furtherprocessing.

As shown in FIGS. 9A-9C, the tissue chamber 509 and or the spring-loadedplatform 507 may comprise locating posts, tabs, or other members 519 andcomplimentary recesses for accepting said locating tabs, posts, or othermembers. The locating members 519 and corresponding recesses can serveto locate the tissue chamber 509 on the spring-loaded platform 507relative to the inner and outer portions thereof and also relative tothe wax-cutting blades 517 and the plunger 523. Wax-retention members521 (e.g., posts or barbs) are also shown in FIGS. 9A-9C as part of thetissue chamber 509. The wax-retention members 521 are operable to holdand locate the wax-embedded sample 511 within the tissue chamber 509during operation of the wax removal device 501.

FIG. 9A shows the wax removal device 501 in the open position with atissue chamber 509 loaded on the spring-loaded platform 507. FIG. 9Bshows the wax removal device 501 in the wax cutting position where thestops 505 are acting on the central portion 513 and the wax-cuttingblades 517 have cut the wax surrounding the wax-embedded sample 511 butthe plunger 523 has not broken the frangible area of the tissue chamber509. FIG. 9C shows the wax removal device 501 in the wax removalposition where the outside portion 515 has forced the plunger 523downward, breaking the remainder 525 of the tissue chamber downward onthe spring-loaded platform 507 and apart from the now separatewax-embedded sample 511.

FIG. 10 illustrates a container 1001 including a support sponge 1013according to certain embodiments. The container 1001 includes a specimenchamber 1005 to receive a tissue sample as well as two fluid ports 1007to introduce and remove fluid from the specimen chamber 1005. Thecontainer 1001 includes a cover 1003 than encloses the specimen chamber1005 after a tissue sample has been placed therein. The fluid ports 1007may be self-sealing, especially where the cover 1003 is operable form afluid and air tight seal with the top of the container 1001 to create asealed environment within the specimen chamber 1005. As noted above,self-sealing fluid ports 1007 may include a rubberized or silicone plugor surface that permits introduction of a needle but which seals uponneedle removal. In some embodiments the self-sealing ports areneedle-free connectors, such as those that include a self-closing valvethat opens when a tube connector is attached. The container 1001 mayinclude a bottom cover 1015 with a sponge support 1013 or other supportas discussed herein. The sponge support 1013 and/or the bottom cover1015 may form the bottom of the specimen chamber 1005 and may comprisean optically transmissive, transparent, or index-matched material (e.g.,having approximately the same refractive index as the cleared tissuesample to be imaged) such as a optically transmissive window 1011 in thebottom cover 1015.

A sponge or other porous compressible material is configured to contactthe tissue sample and hold the tissue sample in place after positioningwithin a tissue chamber for chemical processing, clearing, and/orimaging. In some embodiments the porous compressible material is aplastic sponge. The sponge cell size may be any size that enablesadequate tissue support with minimal compression and may be anywhere inthe range of 10 μm to 5 mm. The sponge cell size may be in a range thathelps wet both the tissue and optical surfaces without air bubbletrapping. In preferred embodiments, the sponge cell size is between 50and 500 μm when dry. In other preferred embodiments the sponge cell sizeis between 50 and 200 μm. The sponge may be open cell or closed cell. Inpreferred embodiments the sponge is open cell. In preferred embodimentsthe sponge is substantially non-fluorescent. In some embodiments thesponge is fabricated from a material that has a refractive index betweenabout 1.45 and about 1.7. In some embodiments the sponge has arefractive index of between about 1.53 and 1.60. The sponge material maybe selected to approximately match the refractive index of the clearedtissue sample to be imaged. The sponge can comprise a material that isresistant to acid, a material that is resistant to organic solvents suchas BABB, a material that is resistant to alcohols and/or a material thatis resistant to temperatures up to about 75 degrees Celsius.

FIG. 11 illustrates some of the internal configuration of the container1001 shown in FIG. 10 including the internal fluid passages 1017 leadingfrom the fluid ports 1007 to the specimen chamber 1005. The fluid ports1007 is optionally positioned at a planar level offset from the level ofthe specimen chamber 1005, such that they can be oriented higher thanthe specimen chamber 1005 during fluid exchange. Due to the lowerdensity of air relative to processing fluids, such orientation aids inthe removal of air from the specimen chamber 1005 during fluid exchange,ensuring optimal surface contact for dyes and processing chemicals andpreventing imaging distortion due to trapped air.

Methods of the invention may include single-chamber chemical processing,imaging, and wax embedding such that the tissue sample may be initiallypositioned within the chamber in a desired orientation for sectioningand/or imaging and left without further manipulation until removal ofthe wax-embedded sample for sectioning.

Chemical processing may include fixing, dehydrating, clearing, dying,and other steps known in the art and useful for both intact tissueimaging (e.g. fluorescent staining and imaging) and histologicalanalysis (e.g., wax embedding and microtome sectioning). In certainembodiments, the tissue sample may be exposed to one or more stains,fixatives, dehydrants, and/or clearing agents within a single tissuechamber as described herein. In some instances, one or more of the abovestains, fixatives, dehydrants, and/or clearing agents may be combined ina single solution. Suitable examples of chemical processing solutionsand techniques are described in U.S. Pub. 2016/0003716 and U.S. Pub.20160003715, the contents of each of which are incorporated herein byreference.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A method for analyzing a tissue sample, themethod comprising: orienting a tissue sample in a tissue chamber in adesired position; exposing the tissue sample to a first solution forchemical processing in the desired position in the tissue chamber;immersing the chemically processed tissue sample in a fluid in thedesired position in the tissue chamber; and imaging the immersed tissuesample in the desired position in the tissue chamber withoutrepositioning the tissue sample after orienting.
 2. The method of claim1, further comprising wax embedding the tissue sample in the desiredposition in the tissue chamber.
 3. The method of claim 2, wherein thedesired position is a desired position for sectioning of thewax-embedded tissue sample.
 4. The method of claim 1, further comprisingsealing the tissue sample in the tissue chamber after orienting whereinthe first solution and the fluid are introduced to the tissue chamberthrough a sealable port.
 5. The method of claim 1, wherein the fluidcomprises a clearing agent.
 6. The method of claim 5, wherein theclearing agent is BABB.
 7. The method of claim 1, wherein the firstsolution comprises a dehydrant.
 8. The method of claim 1, wherein thefirst solution comprises a fixative.
 9. The method of claim 8, whereinthe fixative is a dehydrant.
 10. The method of claim 1, wherein thefirst solution comprises a dye.
 11. The method of claim 10, wherein thedye is a fluorescent dye and the imaging step comprises fluorescentimaging.
 12. The method of claim 1, wherein the desired position is adesired position for imaging of the tissue sample.
 13. The method ofclaim 1, wherein the tissue chamber comprises a plurality of featuresdisposed on an interior surface of the tissue chamber and configured tocontact the tissue sample and permit fluid flow between the tissuesample and the interior surface.